Erythropoietin (“EPO”) is a glycoprotein that is the primary regulator of erythropoiesis. Specifically, EPO is responsible for promoting the growth, differentiation and survival of erythroid progenitors, which give rise to mature red blood cells. In response to changes in the level of oxygen in the blood and tissues, erythropoietin appears to stimulate both proliferation and differentiation of immature erythroblasts. It also functions as a growth factor, stimulating the mitotic activity of erythroid progenitor cells, such as erythrocyte burst forming and colony-forming units. It also acts as a differentiation factor, triggering transformation of an erythrocyte colony-forming-unit into a proerythroblast (See Erslev, A., New Eng. J. Med., 316:101-103 (1987)).
EPO has a molecular weight of about 34,000 daltons and can occur in three forms—alpha, beta and asialo. During mid- to late gestation, EPO is synthesized in the fetal liver. Subsequently, EPO is synthesized in the kidney, circulates in the plasma and is excreted in the urine.
Human urinary EPO has been isolated and purified (See, Miyake et al., J. Biol. Chem., 252:5558 (1977)). Moreover, methods for identifying, cloning and expressing genes encoding EPO (See U.S. Pat. No. 4,703,008) as well as purifying recombinant EPO from a cell medium (See U.S. Pat. No. 4,667,016) are known in the art.
The activity of EPO is mediated through the binding and activation of a cell surface receptor referred to as the erythropoietin receptor. The EPO receptor belongs to the cytokine receptor superfamily and is believed to contain at least two distinct polypeptides, a 55-72 kDa species and a 85-100 kDa species (See U.S. Pat. No. 6,319,499, Mayeux et al., J. Biol. Chem, 266:23380 (1991), McCaffery et al., J. Biol. Chem., 264:10507 (1991)). Other studies have revealed other polypeptide complexes of EPO receptor having molecular weights such as 110, 130 and 145 kDa (See U.S. Pat. No. 6,319,499).
Both the murine and human EPO receptors have been cloned and expressed (See D'Andrea et al., Cell, 57:277 (1989); Jones et al., Blood, 76:31 (1990); Winkelmann et al., Blood, 76:24 (1990); WO 90/08822/U.S. Pat. No. 5,278,065). The full length human EPO receptor is a 483 amino acid transmembrane protein with an approximately 25 amino acid signal peptide (See U.S. Pat. No. 6,319,499). The human receptor demonstrates about a 82% amino acid sequence homology with the murine receptor. Id.
In the absence of ligand the EPO receptor exists in a preformed dimer. The binding of EPO to its receptor causes a conformational change such that the cytoplasmic domains are placed in close proximity. While not completely understood, it is believed that this “dimerization” plays a role in the activation of the receptor. The activation of the EPO receptor results in a number of biological effects. Some of these activities include stimulation of proliferation, stimulation of differentiation and inhibition of apoptosis (See U.S. Pat. No. 6,319,499, Liboi et al., PNAS USA, 90:11351 (1993), Koury, Science, 248:378 (1990)).
It is the relationship between the EPO receptor dimerization and activation that can be used to identify compounds (i.e. such as antibodies) other than EPO that are capable of: (1) dimerizing the EPO receptor; and (2) activating the receptor. These compounds would be useful in treating mammals suffering from anemia and in identifying mammals having a dysfunctional EPO receptor.